1 | INTRODUCTION
Cytosolic protein delivery promisingly expands therapeutic possibilities[1,2]. The delivery of active proteins replaces disease-causing deficient or dysfunctional proteins that are important for essential cellular events[3]. Recently, the delivery of engineered proteins, such as CRISPR/Cas9, has been reported to artificially modulate genomic information, thereby opening a new window for therapeutic applications[4]. However, the development of precise and efficient technologies for cytosolic protein delivery remains challenging. As proteins are generally membrane-impermeable owing to their macromolecular nature and hydrophilic properties, most proteins cannot spontaneously enter the cells through cell membranes. Cell-permeable carriers consisting of peptides[5], polymers[4,6-8], liposomes[9,10], and nanoparticles[11] have been actively employed to transport proteins inside cells. Although a few carriers have achieved direct cytosolic delivery of proteins by fusion with cellular plasma membranes[10,11], most conventional carriers are usually taken up via endocytosis. Without being released from endosomes, cargo proteins are degraded in lysosomal compartments before being functional in the cytosol[2]. Therefore, technologies for endosomal escape have been developed by utilizing the proton sponge effect of pH-buffering agents, such as poly(ethyleneimine)[12] and a charge conversion block polymer[6], by fusion of a carrier with the endosomal membrane[13] and by destabilization of the endosomal membrane with endosome-disruptive peptides[14,15], polymers[7,16], nanoparticles[11,17], and photosensitizers[18-21]. Among these technologies, photochemical approaches offer photo-triggered spatiotemporal delivery of cargo proteins into the cytosol[19-22]. Selective cytosolic protein delivery at the desired timing and sites holds promise for safer and more effective therapies[1,22]. However, the light used during most photochemical reactions generally does not penetrate through the deeper areas of the body because of its low permeability in living tissues[23].
Ultrasound readily penetrates deep into the interior of the body in a noninvasive manner [24]. Using focusing techniques, ultrasound exposure can be limited to a localized region, leading to selective exposure at the target site. Accordingly, ultrasound-responsive carriers for protein delivery have been reported to be promising non-invasive tools for spatiotemporal administration of proteins in deeper areas of the body[25-27]. However, no ultrasound-responsive carriers for cytosolic protein delivery have been reported till date. A method for ultrasound-induced drug release from endosomes has recently been reported[28]. In a pioneering study, liposomes containing fluorescent dyes and perfluorocarbon nano-droplets (phase-change nano-droplets, PCNDs) were introduced into living cells by folate-mediated endocytosis, and subsequent ultrasound-induced vaporization of PCNDs led to endosome rupture, thereby achieving escape of the dyes from the endosomes. In this study, we envisioned that endosomal rupture through vaporization of PCNDs could be applied to ultrasound-induced cytosolic protein delivery. In our previous reports, PCNDs conjugated with an anti-epiregulin (EREG) antibody (named as 9E5) were taken up by high-EREG-expressing cancer cells via endocytosis[29] and were confirmed to vaporize inside the cells by exposure to ultrasound[30,31]. Therefore, 9E5-conjugated PCNDs were used as carriers for cytosolic protein delivery. Cargo proteins were conjugated with PCNDs via a bio-reductively cleavable disulfide linker. Protein-conjugated PCNDs were introduced into living cells via 9E5-mediated endocytosis, and after exposure to ultrasound, the cytosolic delivery of an enzyme and a cytotoxic protein was examined (Figure 1).